Fractionation of DNA nucleotide transcripts from Moloney sarcoma virus and isolation of sarcoma virus-specific complementary DNA

Abstract

Radioactive DNA complementary to nucleotide sequences in Moloney murine sarcoma virus (MSV) and Moloney leukemia virus (M-MuLV) complex was made by the endogenous reverse transcriptase reaction. These virus stocks contained a threefold excess of MSV over M-MuLV as measured by biological assay. The complementary DNA was an accurate copy of the viral RNA in that 86% of 35S viral RNA hybridized with complementary (cDNA) DNA at a 1.5 to 1 cDNA-RNA molar ratio. The complementary DNA, of a 4-6S size, was fractionated by sequential absorptions with MulV and the feline leukemia virus pseudotype of MSV, [MSV(FeLV)] RNA. In this manner three sets of nucleotide sequences whichrepresent different portions of the MSV viral complex were obtained: a sarcoma virus-specific fraction (cDNAsarc) with sequences that had no homology to M-MuLV RNA but which hybridized to MSV (FeLV) RNA, a sarcoma-leukemia fraction (cDNA common) with sequences common to MSV as well as M-MuLV viral RNA, and a cDNAleuk representing those nucleotide sequences found only in M-MuLV. Hybridization of MSV-MuLV viral 35S RNA with a threefold molar excess of cDNA's revealed that approximately 20% was hybridized with cDNAsarc, whereas approximately 75% was hybridized with cDNAcommon. M-MuLV 35S RNA alone did not hybridize with cDNAsarc but did hybridize 40 and 50% with cDNAleuk and cDNAcommon, respectively. The cDNAsarc represents about 25% of the total MSV sequences, whereas the cDNAcommon represents the remainder of the MSV virus genome. Some cDNAcommon sequences were shared by two other sarcoma viruses and several distinctly different isolates of MulV. In contrast, the MSV "sarc" sequences had little or no homology with two other murine sarcoma virus isolates.